ETS is a multigene family, and its members share a common ETS DNA-binding domain. ETS family genes encode transcription factors that recognize the GGAA core sequence and activate transcription via binding to these sequences. By searching the sequence data base, more than 100 cellular and viral promoters/enhancers that contain either single, dual (palindromic or direct repeat) or multiple Ets-binding sites (EBS) have been identified. To identify the functional ones, DNA binding and transactivation assays were carried out using several single and dual sites. Here, it is reported that the ETS1, ETS2 and ERGB/Hu-FLI-1 proteins transactivate transcription via binding to the single sites located in the promoter/enhancers of N-ras, GADD153, HIV-2 LTR, IL4, 2'-5' OAS, G-CSF, and dual sites located in HIV-1 LTR, p53, GATA-1 and MHC class I genes. Although the ETS proteins bind to and activate transcription from the single sites, the flanking nucleotides display strict requirements that influence binding efficiency and transactivation activity. It appears that the PuPyPu sequence at the 3'-end of the GGAA core is essential for binding and transactivation and an efficient single site seems to have a sequence of either CA/CGGAAPuPyPu or GCGGAAPuPyPu. ETS1 DNA binding on dual sites (p53, HIV-1 LTR and GATA-1) is dramatically increased in the presence of a monoclonal antibody whose epitope maps between amino acid positions 240-260 (Seth et al., 1993, Oncogene, in press). Transactivation and DNA binding data suggest that the two EBS's within a dual site cooperate in binding activity and the nucleotides flanking the GGAA core in dual sites could be different than those flanking the GGAA core in single sites.